François Briand,1 Eric Mayoux,2 Emmanuel Brousseau,1 Noémie Burr,1

Isabelle Urbain,1 Clément Costard,1 Michael Mark,2 and Thierry Sulpice1

Empagliflozin, via SwitchingMetabolism Toward Lipid Utilization,Moderately Increases LDL CholesterolLevels Through Reduced LDLCatabolismDiabetes 2016;65:2032–2038 | DOI: 10.2337/db16-0049

In clinical trials, a small increase in LDL cholesterol hasbeen reported with sodium–glucose cotransporter 2(SGLT2) inhibitors. The mechanisms by which the SGLT2 in-hibitor empagliflozin increases LDL cholesterol levels wereinvestigated in hamsters with diet-induced dyslipidemia.Compared with vehicle, empagliflozin 30 mg/kg/day for2 weeks significantly reduced fasting blood glucose by18%, with significant increase in fasting plasma LDLcholesterol, free fatty acids, and total ketone bodies by25, 49, and 116%, respectively. In fasting conditions,glycogen hepatic levels were further reduced by 84%withempagliflozin, while 3-hydroxy-3-methylglutaryl-CoAreductase activity and total cholesterol hepatic levelswere 31 and 10% higher, respectively (both P < 0.05 vs.vehicle). A significant 20% reduction in hepatic LDLreceptor protein expression was also observed withempagliflozin. Importantly, none of these parameters werechanged by empagliflozin in fed conditions. Empagliflozinsignificantly reduced the catabolism of 3H-cholesteryloleate–labeled LDL injected intravenously by 20%, in-dicating that empagliflozin raises LDL levels throughreduced catabolism. Unexpectedly, empagliflozin also re-duced intestinal cholesterol absorption in vivo, which ledto a significant increase in LDL- andmacrophage-derivedcholesterol fecal excretion (both P < 0.05 vs. vehicle).These data suggest that empagliflozin, by switchingenergy metabolism from carbohydrate to lipid utilization,moderately increases ketone production and LDL choles-terol levels. Interestingly, empagliflozin also reduces intes-tinal cholesterol absorption, which in turn promotesLDL- and macrophage-derived cholesterol fecal excretion.

Specific sodium glucose cotransporter (SGLT) inhibitorsrepresent an emerging and promising new class of glucose-lowering drugs in the management of type 2 diabetes.The unique mode of action of this class of novel agentscan effectively decrease blood glucose levels, independentlyof the insulin pathway, via increasing glucose excretion inurine, i.e., glucosuria (1,2). Besides improved glycemic pa-rameters, SGLT2 inhibitors have shown additional benefitssuch as body weight loss and blood pressure–lowering, withlow risk of hypoglycemia (3). However, an increase in LDLcholesterol (LDL-C) plasma levels has also been observed inpatients treated with SGLT2 inhibitors (1). The mechanismby which SGLT2 inhibition raises LDL-C levels remainsunclear. It has been suggested that the increase in LDL-Cmay be partly due to hemoconcentration, as SGLT2 inhib-itors induce volume contraction subsequent to increasedurinary volume (4,5). However, the transient diuretic effectof SGLT2 inhibitors may not completely contribute tothe observed LDL-C increase. We therefore investigatedthe effects of the SGLT2 inhibitor empagliflozin in thediet-induced insulin-resistant dyslipidemic golden Syrianhamster, a validated preclinical model with cholesterolmetabolism similar to that of humans (6,7).

RESEARCH DESIGN AND METHODS

All animal protocols were approved by the local (Comitérégional d’éthique de Midi-Pyrénées) and national (Ministèrede l’Enseignement Supérieur et de la Recherche) ethicscommittees. Male golden Syrian hamsters (91–100 g,

1Physiogenex SAS, Prologue Biotech, Labège, France2Cardiometabolic Diseases Research, Boehringer Ingelheim, Biberach an derRiss, Germany

Corresponding author: François Briand, f.briand@physiogenex.com.

Received 11 January 2016 and accepted 31 March 2016.

F.B. and E.M. contributed equally to this study.

© 2016 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, andthe work is not altered.

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PHARMACOLOGYAND

THERAPEUTIC

S

6 weeks old; Elevage Janvier, Le Genest Saint Isle, France)were fed ad libitum over 4 weeks with a high-fat/high-cholesterol diet (0.5% cholesterol, 0.25% deoxycholate,11.5% coconut oil, and 11.5% corn oil) with 10% fructosein the drinking water as previously described (7). After2 weeks of diet to induce dyslipidemia, hamsters wererandomized into two sets of nonradioactive (set 1) orradioactive (set 2) experiments, according to blood glu-cose and LDL-C levels in fed or overnight fasting condi-tions (fasting starting at 5:00 P.M. and blood collection at;8:00 A.M.), and were then treated orally for 2 weekswith vehicle or empagliflozin 30 mg/kg once daily. Thedose was selected from a pilot study where glucose urine ex-cretion was measured in this hamster model treated acutelywith empagliflozin 3, 10, and 30 mg/kg. The 30 mg/kgdose was found to increase glucose urine excretion by1,200-fold versus vehicle, while the 3 and 10 mg/kg dosesshowed a slighter effect (80- and 200-fold, respectively). Atthe end of the treatment period, a first set of hamsterswas used to measure biochemical parameters using com-mercial kits in fed or overnight fasting conditions. Lipo-protein total cholesterol profile was assessed using fastprotein liquid chromatography analysis using one pooledplasma sample (one pool per treatment group); Westernblot analyses for LDL receptor protein expression and fecalcholesterol mass excretion were performed as previouslydescribed (7). A second set of hamsters underwent radio-active tracer–based in vivo experiments to measure in-testinal cholesterol absorption, LDL cholesteryl esters

kinetics, or macrophage-to-feces reverse cholesterol transportas previously described (6,7). Intestinal cholesterol absorp-tion was assessed after administration of 14C-cholesterol–labeled olive oil by oral gavage and intraperitoneal injectionof poloxamer-407 (a lipase inhibitor) to measure 14C-tracerplasma tracer appearance at time 3, 5, and 6 h after oral ga-vage (6). Kinetics of LDL cholesteryl oleate were performedby intravenously injecting 3H-cholesteryl oleate–labeledLDL in overnight fasted hamsters, previously isolatedfrom hamsters fed the same high-fat/high-cholesteroldiet (7). Hamsters were kept fasted for the first 6 h ofthe kinetic experiment and were then kept in individualcages with access to food and water for feces collectionover 72 h. Plasma 3H-tracer decay curve was monitoredover 72 h after injection to calculate 3H-cholesteryl ole-ate LDL fractional catabolic rate using Simulation Anal-ysis and Modeling (SAAM II) software. Liver (collectedafter 72 h) and feces were used to measure 3H-tracerrecovery in cholesterol and bile acid fraction after chem-ical extraction (6,7).

Macrophage-to-feces reverse cholesterol transport wasmeasured over 72 h after intraperitoneally injecting3H-cholesterol–labeled/oxidized LDL–loaded J774 mac-rophages (6,7). In this experiment, hamsters were notfasted and had constant access to food and water over72 h. Plasma 3H-tracer appearance was measured every24 h, and liver (collected after 72 h) and feces (collectedover 72 h) were used to measure 3H-tracer recovery incholesterol and bile acid fraction after chemical extraction.

Table 1—Body weight and biochemical parameters in fed or overnight fast conditions

Parameters

Fed conditions Overnight fasting conditions

VehicleEmpagliflozin30 mg/kg Vehicle

Empagliflozin30 mg/kg

Body weight (g) 110 6 2 114 6 2 110 6 2 111 6 1

Hematocrit (%) 49.8 6 0.7 47.9 6 0.6* 48.3 6 0.5 49.4 6 0.6

Plasma total protein (g/L) 81.2 6 1.8 81.9 6 1.8 79.6 6 2.5 76.0 6 1.0

Blood glucose (mg/dL) 86.0 6 5.5 88.6 6 2.6 73.4 6 4.0 59.9 6 2.5*

Plasma total cholesterol (g/L) 4.0 6 0.2 4.0 6 0.2 3.0 6 0.1 2.9 6 0.2

Plasma LDL-C (g/L) 1.8 6 0.1 1.6 6 0.1 1.2 6 0.1 1.5 6 0.1*

Plasma ketone bodies (mmol/L) 773 6 76 909 6 124 3,094 6 171 6,685 6 510‡

Plasma free fatty acids (mmol/L) 0.62 6 0.06 0.70 6 0.05 0.45 6 0.03 0.67 6 0.05†

Plasma free glycerol (g/L) 0.023 6 76 0.033 6 0.004* 0.009 6 0.001 0.011 6 0.001

Liver weight (g) 5.61 6 0.13 6.04 6 0.13* 4.90 6 0.13 4.75 6 0.06

Hepatic triglycerides (mg/g liver) 15.1 6 0.9 16.9 6 0.1 16.6 6 1.3 15.3 6 0.7

Hepatic cholesterol (mg/g liver) 38.9 6 0.8 40.2 6 1.7 43.1 6 1.9 47.7 6 1.1*

Hepatic fatty acids (mmol/g liver) 362 6 9 352 6 12 386 6 11 418 6 8*

Hepatic ketone bodies (mmol/g liver) 12.4 6 0.5 12.1 6 0.5 14.7 6 0.6 16.8 6 0.8

Hepatic pyruvate (mmol/g liver) 6.2 6 0.5 6.4 6 0.3 6.7 6 0.4 8.0 6 0.3*

Hepatic HMG-CoAred activity (mU/mg protein) 0.302 6 0.034 0.357 6 0.040 0.255 6 0.019 0.334 6 0.028*

Hepatic glycogen (mg/g liver) 39.1 6 3.9 37.3 6 2.2 4.31 6 0.64 0.7 6 0.4‡

Data are mean 6 SEM. n = 9–10 hamsters/group. HMG-CoAred, HMG-CoA reductase. *P , 0.05 vs. vehicle. †P , 0.01 vs. vehicle.‡P , 0.001 vs. vehicle.

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Data are expressed as mean 6 SEM. Unpaired Stu-dent t test or one-way ANOVA plus Dunnett posttest wasused for statistical analysis. A P , 0.05 was consideredsignificant.

RESULTS

Empagliflozin treatment significantly triggered more bio-chemical parameter changes in the overnight fasting con-dition than in the fed condition (Table 1).

Plasma LDL-C levels were found to be higher by 25% inhamsters treated with empagliflozin (P , 0.05 vs. vehicle)only in the fasting condition. Concomitantly, fasting bloodglucose was reduced by 18% (P , 0.05 vs. vehicle), whileplasma total ketone bodies and free fatty acids were raisedby 116% (P , 0.001 vs. vehicle) and 49% (P , 0.01 vs.vehicle), respectively. Hepatic total cholesterol and fattyacid levels in overnight fasting conditions were 10 and8% higher in hamsters treated with empagliflozin (both

P , 0.05 vs. vehicle). In addition, hepatic total ketonebody levels were 14% higher with empagliflozin, al-though not significantly. Hepatic pyruvate levels and3-hydroxy-3-methylglutaryl (HMG)-CoA reductase activitywere 19 and 31% higher, respectively, in overnight fastedhamsters treated with empagliflozin (both P , 0.05 vs.vehicle). Compared with vehicle, hepatic glycogen levelswere dramatically blunted by 84% with empagliflozin(P , 0.001 vs. vehicle). In sharp contrast with the fastingcondition, empagliflozin showed limited effects on bio-chemical parameters measured in the fed condition withthe exception of minor differences on hematocrit, liverweight, and plasma free glycerol compared with vehicle.

For further confirmation of the raise in plasma LDL-Clevels, total cholesterol lipoprotein profile in overnightfasted hamsters was measured by fast protein liquidchromatography (Fig. 1A). As expected, empagliflozin ledto higher total cholesterol levels in fractions corresponding

Figure 1—Lipoprotein total cholesterol profiles assessed by fast protein liquid chromatography from pooled plasma samples (A), representativeWestern blots and hepatic LDL receptor protein expression after densitometry analysis (B), in vivo intestinal 14C-cholesterol absorption (C), andfecal cholesterol mass excretion (D) in hamsters treated with vehicle or empagliflozin 30 mg/kg/day. *P < 0.05 and ***P < 0.001.n = 9–10 hamsters/group.

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to LDL. Since higher plasma LDL-C may be linked to lowerLDL receptor expression, Western blot analysis was alsoperformed using liver samples collected from overnightfasted hamsters. Compared with vehicle, hepatic proteinexpression of the LDL receptor was found to be reducedby 20% (Fig. 1B) in overnight fasted hamsters treated withempagliflozin (P , 0.05 vs. vehicle).

As higher LDL-C levels could also be related to increasedintestinal cholesterol absorption, this mechanism was alsomeasured in vivo after oral administration of 14C-cholesterol–labeled olive oil. Strikingly, hamsters treated with empagli-flozin showed a 14C-tracer plasma appearance reduced byup to 40% over 6 h after 14C-tracer administration, indi-cating lower intestinal cholesterol absorption (Fig. 1C). In

Figure 2—3H-cholesteryl oleate–labeled LDL plasma decay curve over 72 h and LDL cholesteryl ester fractional catabolic rate (A); 3H-tracerrecoveries in whole liver homogenate, cholesterol, and bile acid fractions (B); and 3H-tracer recoveries in fecal cholesterol and bile acidsfractions (C) at time 72 h after 3H-cholesteryl oleate–labeled LDL intravenous injection. 3H-tracer appearance in plasma over 72 h (D);3H-tracer recoveries in whole liver homogenate, cholesterol, and bile acid fractions (E); and 3H-tracer recoveries in fecal cholesterol and bileacids fractions (F) at time 72 h after 3H-cholesterol–labeled/oxidized LDL–loaded macrophage intraperitoneal injection. Hamsters treatedwith vehicle or empagliflozin 30 mg/kg/day are represented with white bars, open circles, or black dashed bars, closed circles, respectively.*P < 0.05, **P < 0.01, and ***P < 0.001. n = 9–10 hamsters/group.

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agreement with the lower intestinal cholesterol absorption,fecal cholesterol mass excretion was 49% higher in hamsterstreated with empagliflozin (Fig. 1D) compared with vehicle(P , 0.01).

We next investigated LDL-C metabolism in vivo byinjecting 3H-cholesteryl oleate–labeled LDL intravenouslyin hamsters. Empagliflozin treatment resulted in slowed3H-tracer decay curve over 72 h, leading to a 20% reductionin LDL cholesteryl ester catabolism (Fig. 2A), compared withvehicle (P , 0.05). At 72 h after 3H-cholesteryl oleate–labeled LDL, hepatic 3H-tracer recoveries in the whole liverand the hepatic cholesterol fraction were respectively reducedby 11% (P , 0.01 vs. vehicle) and 19% (P , 0.001 vs.vehicle) with empagliflozin treatment (Fig. 2B). As a resultof reduced cholesterol absorption in the intestine, LDL-derived 3H-cholesterol fecal excretion was 26% higher(P , 0.05 vs. vehicle) in hamsters treated with empagli-flozin (Fig. 2C).

For investigation of macrophage-to-feces reverse cho-lesterol transport in vivo, hamsters were injected intraper-itoneally with 3H-cholesterol–labeled/oxidized LDL–loadedmacrophages. Compared with vehicle, empagliflozin did notchange plasma 3H-tracer appearance over 72 h (Fig. 2D).Hepatic 3H-tracer recoveries in the whole liver and thehepatic cholesterol fraction tended to be reduced with

empagliflozin, although this was not significant (Fig. 2E).However, 3H-cholesterol fecal excretion (Fig. 2F) wasincreased by 29% in hamsters treated with empagliflozin(P , 0.05). These data indicate that reduced intestinalcholesterol absorption with empagliflozin treatment pro-motes fecal excretion of cholesterol deriving from themacrophage.

DISCUSSION

The current study indicates that empagliflozin raises LDL-Clevels only in fasting conditions through reduction in LDL-Ccatabolism and alters cholesterol metabolism at both thehepatic and intestinal levels in hamsters.

Overnight fasted hamsters treated with empagliflozinshowed higher LDL-C levels concomitant with higherfree fatty acids and total ketone body plasma levels. Thehigher level of total ketone bodies and fatty acids is inagreement with previous reports indicating that chronictreatment with SGLT2 inhibitors induces ketogenesisand a metabolism switch toward lipid oxidation to counter-balance the carbohydrate restriction in the fasting state(8–10). The excretion of glucose via urine and relatedcalorie loss with SGLT2 inhibition therefore replicatestarvation shift from carbohydrate to lipid utilization forenergy in the fasting state (11). Chronic SGLT2 inhibition

Figure 3—Proposed mechanisms for the alteration of cholesterol metabolism by empagliflozin. SGLT2 inhibition switches from carbohy-drate to fat oxidation and stimulates ketone body production and hepatic cholesterol synthesis in fasting conditions. These metabolicalterations result in lower LDL receptor (LDL-r) expression and moderate increase in LDL-C levels. The reduced intestinal cholesterolabsorption, which leads to higher macrophage- and LDL-derived cholesterol fecal excretion, remains to be further investigated. HMGCS1,HMG-CoA synthase 1; HMGCS2, HMG-CoA synthase 2; HMGCoA red, HMG-CoA reductase.

2036 SGLT2 Inhibition and LDL-C Diabetes Volume 65, July 2016

also seems to mimic the LDL-raising effects of ketogenicdiet, in which LDL-C levels correlate with blood ketonebody levels (12). In the current study, evidence for a met-abolic shift toward fat utilization was also observed at theliver level (e.g., hepatic glycogen and pyruvate levels) infasted hamsters treated with empagliflozin. The increasedhepatic fatty acids levels may fuel the pool of acetyl-CoA,an important metabolic branch point, as a source for bothketone body production and hepatic cholesterol synthesis(13), with the latter associated with higher HMG-CoAreductase activity and hepatic total cholesterol levels.As hepatic levels of cholesterol regulate LDL receptorexpression (14,15), empagliflozin treatment lowered LDLreceptor expression and plasma LDL-C catabolism, whichin turn increased LDL-C plasma levels. Although a raise inLDL-C levels is seen as an increase in cardiovascular eventrisk (16), it is probably not so prominent with empagliflozin.Indeed, the EMPA-REG OUTCOME study (BI 10773[Empagliflozin] Cardiovascular Outcome Event Trial inType 2 Diabetes Mellitus Patients) recently delivered aspectacular 38% reduction in cardiovascular mortalityand 35% reduction in hospitalization with heart failure,with no change in event rate of nonfatal myocardial in-farction and nonfatal stroke (17). Moreover, our studyrevealed that even after chronic treatment with empagli-flozin, the increase in LDL-C was only observed in theovernight fasted condition. In the clinical setting, LDL-Clevels are routinely assessed from plasma collected in thefasted state. Therefore, clinical investigations evaluat-ing the effects of empagliflozin on LDL-C levels in fedconditions would be of interest. In addition, our in vivoexperiments also highlighted potential antiatherogenicmechanisms induced by empagliflozin, such as LDL-and macrophage-derived fecal cholesterol excretion.Macrophage-to-feces reverse cholesterol transport is knownto be inversely correlated with atherosclerosis (18), andan enhanced excretion of LDL-derived cholesterol inthe feces theoretically prevents its accumulation inthe arterial wall. Whether these mechanisms, besidesbody weight loss and blood pressure lowering, con-tribute to the reduced cardiovascular risk in patientstreated with empagliflozin (17) remains to be furtherinvestigated.

Another point of investigation is the reduced intestinalcholesterol absorption observed in hamsters treated withempagliflozin. Since a balance exists between hepaticcholesterol synthesis and intestinal cholesterol absorption(19), the lower intestinal cholesterol absorption may there-fore result from the stimulation of hepatic cholesterol syn-thesis by empagliflozin. However, the molecular mechanismby which empagliflozin alters intestinal cholesterol metabo-lism remains to be elucidated.

In conclusion, the current study suggests that empagli-flozin raises LDL-C levels only in the fasting condition byreducing LDL receptor expression and LDL-C catabolism.As illustrated in Fig. 3, the proposed mechanism leadingto the LDL-C increase originates from the metabolic

switch toward lipid utilization, which triggers in parallela moderate activation of ketogenesis pathway and hepaticcholesterol synthesis within the liver. Future studies totest whether SGLT2 inhibitors have a similar rhythmiceffect in plasma from patients fasted versus fed would berequired.

Acknowledgments. The authors thank Dominique Lopes for animal care,Marjolaine Quinsat and Hélène Lakehal for technical assistance, and AurélieCouderc for quality control (all of Physiogenex).Duality of Interest. This work has been funded by Boehringer Ingelheim.E.M. and M.M. are employees of Boehringer Ingelheim. F.B., E.B., N.B., I.U., C.C.,and T.S. are employees of Physiogenex.Author Contributions. F.B., E.M., M.M., and T.S. designed research.F.B., E.B., N.B., I.U., and C.C. conducted research. F.B. and E.M. analyzed dataand wrote the manuscript. T.S. had the primary responsibility for the finalcontent. All authors read and approved the final manuscript. T.S. is theguarantor of this work and, as such, had full access to all the data in the studyand takes responsibility for the integrity of the data and the accuracy of thedata analysis.Prior Presentation. Parts of this study were presented in abstract form atthe 51st Annual Meeting of the European Association for the Study of Diabetes,Stockholm, Sweden, 14–18 September 2015.

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17. Zinman B, Wanner C, Lachin JM, et al.; EMPA-REG OUTCOME Investigators.Empagliflozin, cardiovascular outcomes, and mortality in type 2 diabetes. N EnglJ Med 2015;373:2117–212818. Rader DJ, Alexander ET, Weibel GL, Billheimer J, Rothblat GH. The role ofreverse cholesterol transport in animals and humans and relationship to ath-erosclerosis. J Lipid Res 2009;50(Suppl.):S189–S19419. Miettinen TA, Gylling H, Viikari J, Lehtimäki T, Raitakari OT. Synthesis andabsorption of cholesterol in Finnish boys by serum non-cholesterol sterols: thecardiovascular risk in Young Finns Study. Atherosclerosis 2008;200:177–183

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